Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast a-factor cDNA and expressed in the Pichia pastoris yeast expression system. Fulllength elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fastacting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k ass values of 2-4 · 10 6 M )1

Neutrophil proteinase‐mediated lung tissue destruction is prevented by inhibitors, including elafin and its precursor, trappin. We wanted to establish whether neutrophil‐derived oxidants might impair the inhibitory function of these molecules. Myeloperoxidase/H2O2 and N‐chlorosuccinimide oxidation of the inhibitors was checked by mass spectrometry and enzymatic methods. Oxidation significantly lowers the affinities of the two inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This decrease in affinity is essentially caused by an increase in the rate of inhibitory complex dissociation. Oxidized elafin and trappin have, however, reasonable affinities for NE (Ki = 4.0–9.2 × 10−9 m) and for Pr3 (Ki = 2.5–5.0 × 10−8 m). These affinities are theoretically sufficient to allow the oxidized inhibitors to form tight binding complexes with NE and Pr3 in lung secretions where their physiological concentrations are in the micromolar range. Yet, they are unable to efficiently inhibit the elastolytic activity of the two enzymes. At their physiological concentration, fully oxidized elafin and trappin do not inhibit more than 30% of an equimolar concentration of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin strongly impairs their activity. Inhibitor‐based therapy of inflammatory lung diseases must be carried out using oxidation‐resistant variants of these molecules.